Project 2 Summary/Abstract Mycobacterium tuberculosis (Mtb) infects over 2 billion people worldwide and causes 1.4 million deaths annually. The standard treatment for tuberculosis (TB) due to drug-susceptible (DS) Mtb consists of 2 months of rifampin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) followed by 4 months of RIF and INH. Emergence of drug resistance in Mtb is due, in part, to patient non-compliance with the 6 month regimen. In human TB, Mtb exists in 3 metabolic states: log phase growth (LPG), semi-dormant acidic phase growth (APG), and a non-replicating persister (NRP) state. RIF, INH and EMB kill LPG Mtb while PZA kills APG Mtb. RIF also kills NRP Mtb. Thus, only one drug in the standard regimen is active against APG and NRP Mtb. In studies in which new drugs with novel mechanisms of action were added to the FDA-approved antibiotics for the treatment of DS-Mtb and multdrug- resistant TB, the time to bacterial sterilization in animal models and for converting sputum cultures to negative in clinical trials were shortened, showing that regimens consisting of FDA-approved first- and second-line TB drugs are not optimized to kill Mtb. Our long term objective is to develop highly effective TB regimens. The overarching hypothesis is that TB regimens that are pharmaco- dynamically (PD) optimized to rapidly kill Mtb in all 3 metabolic states will provide potent short- course TB regimens that will maximize disease cure and prevent emergence resistance. We will test this hypothesis and develop potent short-course TB regimens by completing the following Specific Aims (SAs): SA #1: Using our novel murine TB model in which the effect of drugs on the killing of Mtb in the 3 metabolic states is quantifiable, delineate the serum and lung epithelial lining fluid (ELF) pharmacokinetics (PKs) of 5 new TB drugs with differing activities against Mtb in the 3 metabolic states. The lung ELF PK profiles will be simulated in the in vitro hollow fiber PD studies in Project 1 and will be central to translating the in vitro hollow fiber and animal study results to highly effective, short-course TB regimens for man. SA #2: Using our novel murine TB model, conduct 2-month dose-range studies with the 5 new TB drugs as mono-agents to identify the lowest dose of each drug that maximizes the killing of LPG/APG and NRP Mtb and prevents resistance. SA #3: Using our novel murine TB model, conduct 2-month efficacy studies for the 2-drug regimens that are most active in killing Mtb in each metabolic state in the in vitro checkerboard drug-interaction studies (Project 1) and the murine dose-range studies (SA #2). Quantify the extent to which the drugs in combination (a) are synergistic, (b) kill LPG/APG and NRP Mtb and (c) prevent resistance. SA #4: It is possible that the individual 2-drug regimens examined in SA #3 do not maximally kill Mtb in all 3 metabolic states or may not fully prevent resistance. SA #4 will define the efficacy of the sequential use of two 1-month long 2-drug regimens in the killing of Mtb in the 3 metabolic states and for resistance prevention. The drugs in the sequential regimens will have different mechanisms of action to prevent cross-resistance. After completing these SAs we will mathematically derive highly potent, short-course regimens that will effectively treat human TB and prevent resistance. Relevance. This project will use pharmacodynamic methods to generate highly effect, short-course TB drug that will maximize rate and extent of killing of Mtb in the 3 metabolic states and prevent emergence of drug resistance.